Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

5) Page 4. 'separation of ter foci occurred just before cell division' What do you mean with 'cell division'?The onset of constriction, or cell separation (birth)?These events are likely at least 20 min apart under these growth conditions.6) Page 9.The authors note 'that the time between ori duplication and ter centralization is decreased in both Ter-linkage mutants compared to wild-type', but this is difficult to infer from the figures.a) How much shorter?Perhaps a table with relevant numbers can be added?b) Do ori and/or ter foci move faster, or is the period between ori duplication and ori movement shortened?c) Why would a defective Ter-linkage have this effect?7) Page 9.The authors conclude ' that the migration of ter to mid-cell, and its maintenance there, does not require the linkage of the terminus region to the divisome '.However, they ignore the fact that, besides the Ter-linkage, FtsK also interact with the terminus region.Though FtsK activity may partly depend on the Ter-linkage, I don't believe it has been ruled out that FtsK can still bind ter, and perhaps help direct its location, in the absence of a functional Ter-linkage.If it has, please enlighten the reader.a) So, this statement seems too broad, and I'd recommend rewriting it (see also point 2 above).b) It also raises the interesting and relevant question if FtsK is then perhaps involved in ter centralization in wt and/or Ter-linkage mutant cells.Have the authors performed similar experiments with appropriate ftsK mutants (e.g.lacking the FtsK C-terminus)?8) Pages 10-11.The authors nicely show that artificially increasing the time of sister ori 'cohesion' interferes with normal ter centralization.A converse experiment would be to decrease the sister ori cohesion period, which is observed when cells modestly overproduce TopoIV (Wang_G&D 2008).Would early completion of ori segregation also result in 'early' ter centralization, or would this cause temporal uncoupling of the events?Have the authors considered/tried this? 9) Methods a) Strains RM29 and RH3 are missing from Table S1.b) Table S2.Please provide a proper genotype of plasmids, including origin of replication, antibiotic resistance markers, and relevant (inducible) promoters and fused genes.c) Page 15, medium.No antibiotic is mentioned.So, was antibiotic to select for parB plasmids (Amp?) left out of the growth medium in the pre-culture and the mother-machine?10) Some other points a) Page 7.You mean (Fig. 1D, Fig. S1H) rather than (Fig. 1C, Fig. S1G)?b) Page 9, top.You mean (Fig. S6 E,F,H,I) rather than (Fig. 6 E,F,H,I The manuscript by Sadhir et al. describes a coordinated movement of the replication origin (oriC) and terminus (terC) region of E. coli chromosomes in slow-growth conditions.It is a careful quantitative study that elucidates the dynamics of chromosomes during the cell cycle.The work mostly revisits already-known findings but does it more quantitatively than the earlier studies.The main result, "Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli" (the title of the manuscript), has been reported by Bates and Kleckner (Cell 2005): "Strikingly, concomitant with this transition, ter and the ter-side origin switch positions.[..] ter moves inward toward midcell while the ter-side origin becomes localized to the 1/4 position, symmetrical to its sister at the 3/4 position."The most interesting/novel finding from this work is that without splitting of oriC-proximal focus, the Ter still moves to midcell although only for short period of time.It is then instead localizing to the nucleoid periphery in cells where the ori-foci remain at the midcell.The motion to the midcell appears to be driven by DNA replication.I would have expected some mechanistic insights or modeling arising from the data to be included.

Main points of criticism:
1) There is no mechanistic interpretation of findings, which mostly have been known before.Without model the impact of the work will be modest.
2) Title and the section "ter centralisation is coupled to, and requires, ori segregation" in Discussion.When ori region is crosslinked by CFP-ParB, ter centralization still occurs, although for a short period.It seems to me to contradict the statement that ori segregation is required for the ter centralization.
3) The aligned and averaged kymograph in Fig. 4 show vary rapid movement of the Ter focus.Such representation overlooks that Ter can frequently overshoot midcell position (Youngren et al GenesDev 2014), that the focus can split and that in individual cells the movement of Ter focus across the nucleoid can be observed.In other words, the movement of the Ter from the new pole side to nucleoid middle takes much more time then this kymograph shows.The alignment procedure in Fig. 4 is somewhat arbitrary.Why needs the Ter focus to be stable for three consecutive frames?What is the outcome if different criteria are used?
Minor points of criticism: 1) "While in some bacteria this can be directly attributed to the well-studied ParABS partitioning system, in other species this system is either not strictly essential or is absent altogether."-Some references needed.
2) "Here, we use high-throughput single-cell imaging and analysis to quantitatively establish the choreography.." choreography -> movement 3) "Indeed, and surprisingly given the slow growth rate, we found that 15% of cells were born with more than one ori focus (Fig. S1F), indicating that DNA replication was initiated in the previous cell cycle."-This should not be surprising.See S. Tiruvadi-Krishanan et al Cell Reports 2022 and P. Kar et al PNAS 2023.4) "It was recently suggested that ter centralisation shortly precedes the stable appearance of a constricted/bilobed nucleoid structure (Männik et al., 2016)" -This is misquotation of Männik et al., 2016, which states "The permanent constrictions, which were present uninterrupted until cells divided, appeared in all strains only after the Ter region centralized (Figure 5C)."The transient nucleoid constrictions were seen before Ter centralization.Please correct the whole paragraph.5) "Overall, these results confirm that the migration of ter to mid-cell, and its maintenance there, does not require the linkage of the terminus region to the divisome" -This was also shown in Mannik et al 2016.
6) Section "Quantitative timings of cell cycle events" -discussion why timings have been studied instead of cell lengths is more appropriate to the Results or Materials and Method section.7) "Surprisingly, given the mean 133 min cell cycle duration, we found that 15% of cells were born with two ori foci, the majority of which do not initiate replication within their cell cycle (Fig. S1F)."-The argument that the mean doubling time is 133 min in this growth condition is somewhat concerning.It might imply phototoxic effects.Some control with unlabeled strain is needed.
In this growth condition, the majority of cells should have two oris at birth.It is expected that because of noise in the initiation timing, a fraction of these cells "do not initiate replication within their cell cycle" I would have expected some conclusion about the ori-ter dynamics rather than going tangentially to a discussion of double initiations.
Reviewer #3 (Remarks to the Author): Overall impression Sadhir and Murray present an interesting paper addressing the age-old question of how bacterial chromosomes segregate.Experiments are focused primarily on understanding segregation of the terminus region.This is understandable, as ter exhibits dramatic movements and is the only chromosomal locus (in E. coli) known to attach to cell structure capable of directing movement.Positions of ori, ter, and occasionally the nucleoid, are measured in slow growing cells by time lapse imaging in a microfluidics slide.Although there have been (many!) studies of ori and ter dynamics in twenty plus years, their approach is unique in that individual cells are examined over time with high temporal resolution.In addition, they avoid two pitfalls that have plagued many studies: dimeric fluorescent tags and unusual strain backgrounds.Analyses are sophisticated and adhere to a terminus-specific objective throughout the manuscript.They convincingly show that positioning of ter to midcell, an event presumed to be prerequisite for partitioning of daughter chromosomes at division, is independent of MatP-mediated linkage to the division apparatus but dependent on segregation of the origin region.Both of these findings are unexpected and important.Claims that origin segregation is only required for ter positioning (not the bulk nucleoid), and that chromosomes are oriented longitudinally and not transversely, are less convincing.Writing is crisp, and the authors have an uncanny knack for addressing questions and counter points at the moment they occur to the reader.
Major comments 1. Claims that delaying oriC segregation (using a dimeric CFP tag) does not disrupt bulk nucleoid segregation are unsupported by the data.While the effects on ter centralization are clear, there is no examination of nucleoid segregation per se.This is a major point in the paper, addressed in the results and discussion.Segregating nucleoids are even drawn in Figure 6B (although labelled "ori"), as if nucleoids were examined.HU or DAPI imaging should be performed to support this claim.Alternatively, other intermediate loci could be labelled, although this would be unnecessarily difficult unless the tools are already in hand.If possible, showing this data in supplemental would be useful to the field.2. The longitudinal versus transverse debate (if there is one) is not answered by this paper.Like the early FISH studies proposing the longitudinal model with circular chromosome oriented sideways in the cell "ori-arms-ter", data in the current paper are restricted to ori and ter.The transverse model ("left arm-ori-right arm") on the other hand, was born from imaging several arm loci.The fact is, most data examining interstitial loci support the transverse data.I strongly advise softening anti-traverse-model statements in the discussion section "the organization of the chromosome", and instead focusing on how the current data confirms and extends the early ori/ter work and how it fits into the greater evolution of our view of E. coli chromosome organization.
2) As far as FtsK is concerned; most published evidence for its late action in the septal pore applies to its involvement in the Xer recombination step at dif.Whether or not Z-ring associated FtsK can load/unload and translocate (parts of) the chromosome before cell constriction starts is less clear (to this reader at least).The results with the FtsK ATPase mutant by Galli et al does indicate that FtsK translocation is not needed for Ter centralization.However, the Ter-linkage and FtsK could play redundant roles in centralizing and/or in maintaining Ter centralized, and it would be interesting to know if introduction of the FtsK ATPase (or C-terminus) mutant had any effect on Ter dynamics in delta-zapB or delta-matP cells, for example.My thinking may be wrong and, given your modifications of the relevant text, I am not demanding this be done for this paper.But, you do have an excellent set-up to test this and it would be nice to definitively rule out (or in?) a role for FtsK in the Ter centralization step, at some point.
3) Lines 284-285.You speculate that 'the final stage of ori segregation somehow triggers ter to rapidly move to midcell'.Of course, the converse possibility is that ter-centralization somehow forces/signals the ori's to not segregate any (or much) further.Can you explain to the readers why you prefer the first possibility?The response from the authors to my earlier comment that "We did not quote Männik et al.We simply described in our own words the 8 min difference of Männik et al. as "ter centralisation shortly precedes the stable appearance of a constricted/bilobed nucleoid structure"" is not correct.The text of the manuscript (lines 40-42, page 7) includes an explicit citation to Männik et al., 2016 and does not mention 8 min.The authors should be explicitly state that Männik et al. found stable constriction occurred after the Ter centralization (0.06Td (Td-doubling time) based on Fig. 5C in this paper) and they found that stable constriction occurred 45 min after the Ter centralization.Both works show that the stable constriction occurs after the Ter centralization.The author can, of course, state "In contrast" but the comparison should be accurate and not imply that the order of the events they observe is different than in the cited paper.Also, the authors should explain how the timing for stable constriction in nucleoid depends upon the threshold they set.How would it have changed if a higher threshold had been chosen?Why the specific threshold value " …(0.13) is given by the 95th percentile of the relative depth of the nucleoid signal in new-born cells i.e. the first bin of the plot in Figure S3B" was chosen rather than some other criterion?It is very likely that the difference in threshold explains the quantitative difference between the two works more than the growth rate difference, which is invoked now.Reviewer #1 (Remarks to the Author): The authors responded effectively to reviewers comments.High quality and interesting work.I only have a few minor comments.Specific remarks 1) Thanks for the line and page numbers.
2) As far as FtsK is concerned; most published evidence for its late action in the septal pore applies to its involvement in the Xer recombination step at dif.Whether or not Z-ring associated FtsK can load/unload and translocate (parts of) the chromosome before cell constriction starts is less clear (to this reader at least).The results with the FtsK ATPase mutant by Galli et al does indicate that FtsK translocation is not needed for Ter centralization.However, the Ter-linkage and FtsK could play redundant roles in centralizing and/or in maintaining Ter centralized, and it would be interesting to know if introduction of the FtsK ATPase (or C-terminus) mutant had any effect on Ter dynamics in delta-zapB or delta-matP cells, for example.My thinking may be wrong and, given your modifications of the relevant text, I am not demanding this be done for this paper.But, you do have an excellent set-up to test this and it would be nice to definitively rule out (or in?) a role for FtsK in the Ter centralization step, at some point.
Thank you for these comments.It is true that FtsK could have an effect on Ter centralisation/maintenance independent of its ATPase activity and we will certainly consider this in our future experiments.
3) Lines 284-285.You speculate that 'the final stage of ori segregation somehow triggers ter to rapidly move to midcell'.Of course, the converse possibility is that ter-centralization somehow forces/signals the ori's to not segregate any (or much) further.Can you explain to the readers why you prefer the first possibility?This is indeed an alternative possibility.We now explain this and in the discussion we add a sentence explaining that under the hypothesis of entropically-driven rearrangement both schemes are really one and the same thing.4) Line 308.You mean Fig. S7B, F 5C in this paper) and they found that stable constriction occurred 45 min after the Ter centralization.Both works show that the stable constriction occurs after the Ter centralization.The author can, of course, state "In contrast" but the comparison should be accurate and not imply that the order of the events they observe is different than in the cited paper.
We do not imply that the order is different.Could this reviewer be misunderstanding the meaning of the word 'precedes' and the distinction between 'quote' and 'cite'?In any case, we have rephrased the text along the lines the reviewer suggests.Also, the authors should explain how the timing for stable constriction in nucleoid depends upon the threshold they set.How would it have changed if a higher threshold had been chosen?Why the specific threshold value " …(0.13) is given by the 95th percentile of the relative depth of the nucleoid signal in new-born cells i.e. the first bin of the plot in Figure S3B" was chosen rather than some other criterion?It is very likely that the difference in threshold explains the quantitative difference between the two works more than the growth rate difference, which is invoked now.
If we have no minimum threshold (so that any dip in the middle third of the HU profile would count as a constriction), then constriction of the nucleoid would begin ~25 min before Ter centralisation and often even from birth.However, since we do not smoothen the nucleoid profile, this is the result of noise in the signal and is not at all consistent with visual inspection of the images (noise may be more relevant for us than Männik et al 2016 due to the smaller pixel size of our camera (65 nm vs 160 nm)).For example, the cell used in Figure 3A would be marked as constricted from the 5th frame rather than the 19th, while inspection and thresholding of the images shows this is not the case even for the 17th frame (top most cell shown).That is why we use the 95th percentile of cells at birth.Under our conditions, we see no evidence of nucleoid constriction in newborn cells and so we use the 'constriction' depth in these cells as a measure of the noise of the profile.We have now expanded the methods section to make this clear.We presume this reviewer is asking for the frequency at which anucleate cells are produced.We see no significant production of anucleate cells.For the triple labelled strain, we found that ~0.5% of cells born with an inverted organisation produced anucleate daughters.This was comparable to the value (~0.25%) for cells born with a normal organisation.We have now added this to the legend of Fig. S9.
Major point 2, Weak longitudinal model.I agree with the authors that the new data showing left and right arm tags supports the longitudinal model.However, I am curious why they chose not to disclose relative positions of the left and right arm tags, which is provided by their dual labeling approach.As clearly shown in their model (Fig S3L), the relationship between L and R tags is the best test of transverse and longitudinal configurations.A color overlay of S3H (left tag) and S3I (right tag) would do this, with accompanying percentages of observed configurations similar to that shown in Fig S3J/K.Additionally, the figure legend to S3 is somewhat confusing, as some strains contain multiple tags, but individual plots show only one tag.Perhaps the legend and figure labeling could be clarified.
We do not agree that the relationship between L and R tags is the best test of transverse and longitudinal configurations.Given that that nucleoid is not centrally positioned within the cell, one cannot examine, for example, the frequency of L and R being located in opposite cell halves/quarters as that assumes regularly and symmetrically positioned nucleoid lobes.By comparing to the ori and old/new pole as we have done, we can assess the two models using only the ordering of the loci within the cell.What we can do is measure the distance between the loci in the different strains and then compare between them.As shown in the cartoon in S3L, in the longitudinal scheme the loci should be closer to each other than to ori.In the transverse scheme, they should be closer to ori than to each other.We have now added this analysis as well as the requested overlay of S3H and S3I.
We have also clarified the labelling.
Minor points are adequately addressed.
)? c) Legend to Fig. S5c.Incomplete sentence.d) Legend to Fig. S6.Panels H and I are mislabeled G and H. Reviewer #2 (Remarks to the Author):

Reviewer # 3 (
Remarks to the Author): Major point 1, Lack of nucleoid data.Showing DAPI staining (new Fig S9) satisfactorally indicates that chromosome segregation occurred more or less normally in the examples shown.I could not find in the text a reporting of the frequency of normal DAPI segregation.This should be added if absent.Major point 2, Weak longitudinal model.I agree with the authors that the new data showing left and right arm tags supports the longitudinal model.However, I am curious why they chose not to disclose relative positions of the left and right arm tags, which is provided by their dual labeling approach.As clearly shown in their model (Fig S3L), the relationship between L and R tags is the best test of transverse and longitudinal configurations.A color overlay of S3H (left tag) and S3I (right tag) would do this, with accompanying percentages of observed configurations similar to that shown in Fig S3J/K.Additionally, the figure legend to S3 is somewhat confusing, as some strains contain multiple tags, but individual plots show only one tag.Perhaps the legend and figure labeling could be clarified.Minor points are adequately addressed.

Reviewer # 3 (
Remarks to the Author): Major point 1, Lack of nucleoid data.Showing DAPI staining (new Fig S9) satisfactorally indicates that chromosome segregation occurred more or less normally in the examples shown.I could not find in the text a reporting of the frequency of normal DAPI segregation.This should be added if absent.
Sadhir et al. have mostly responded to my comments.Adding some mechanistic interpretation and modeling would have significantly strengthened the manuscript, though.The response from the authors to my earlier comment that "We did not quote Männik et al.We simply described in our own words the 8 min difference of Männik et al. as "ter centralisation shortly precedes the stable appearance of a constricted/bilobed nucleoid structure"" is not correct.The text of the manuscript (lines 40-42, page 7) includes an explicit citation to Männik et al., 2016 and does not mention 8 min.The authors should be explicitly state that Männik et al. found stable constriction occurred after the Ter centralization (0.06Td (Td-doubling time) based on Fig.